Detection of benzimidazole resistance in Haemonchus contortus using RFLP-PCR technique

Benzimidazole (BZ) resistance in Haemonchus contortus is linked primarily with the mutation in the beta-tubulin isotype 1 gene that substitute phenylalanine (Phe) to tyrosine (Tyr) at 200 codon of the gene. In the present study, a new restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique has been developed for detection of BZ resistance in the beta-tubulin isotype 1 gene of H. contortus. The technique utilizes two primers viz. AvikaF and AvikaR to amplify the region containing mutation in the beta-tubulin gene followed by restriction digestion. After digestion, the 'rr' individuals (homozygous resistant) revealed 257 and 48 bp bands, the 'rS' individuals (heterozygous) showed 305, 257 and 48 bp bands, while 'SS' individuals (homozygous susceptible) revealed uncut 305 bp band. A total of 162 adult male H. contortus collected from Avikanagar, Jaipur and Bikaner regions (54 from each region) were genotyped for analyzing BZ resistance in the beta-tubulin gene. Out of which, 130 adults were 'rr' types, 20 'rS' types and 12 'SS' types. The results showed that genotypic frequencies of different genotypes (rr, rS and SS) were highly significant difference among the three regions (P<0.001). The 'rr' individuals were higher (98%) in Jaipur followed by Avikanagar (93%) and Bikaner (50%) regions. Overall, the prevalence of BZ resistant allele (r) was higher (86%) as compared to BZ susceptible allele (S) (14%). The technique was also found suitable for genotyping of larvae of H. contortus and yielded reproducible results. The study indicated that RFLP-PCR is an easy, reproducible and less expensive than allele specific PCR. This technique will be helpful in establishing the prevalence rate of BZ resistance in H. contortus and can also be utilized for existing worm control programme.     

Variability of resistance in goats infected with Haemonchus contortus in Brazil

The variability between and within breeds with respect to nematode egg counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) was studied in 36 yearling female goats of the Caninde (15), Bhuj (6) and Anglo-Nubian (15) breeds, exposed to Haemonchus contortus. Nematode-free goats were turned to a contaminated paddock in late February. From then on, fecal egg per gram counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) were determined at 2-week intervals up to Week 18. The EPG, transformed as [log(EPG + 75)], varied (P < 0.01) between goats within breeds and between weeks of exposure, but not between goat breeds (P > 0.05). PCV and Hb were affected by goat breeds (P < 0.05), by goats within breeds (P < 0.01) and by weeks of exposure (P < 0.01). Anglo-Nubians had higher (P < 0.01) PCV and Hb than Caninde; Bhuj had intermediate values. There were two EPG rises; one between Weeks 6 and 10 and the other between Weeks 14 and 16. The within breed variability was marked during the EPG rise on Week 6, when individual egg counts ranged from 130 to 2500. The EPG rises coincided with drops in Hb. PCV presented a similar trend, though not as marked. Haemonchus was responsible for more than 95% of nematode eggs counted. Considering the goat as experimental unit, the correlation coefficients (r) were: -0.45, P = 0.0064, between log(EPG + 75) and PCV; and -0.53, P = 0.0009, between log(EPG + 75) and Hb. The negative correlation between egg counts and blood values suggested breed differences in PCV and Hb were related to resistance to H. contortus infection and/or to its effects.     

Comparison of three in vitro techniques to estimate benzimidazole resistance in Haemonchus contortus of sheep

Three in vitro assays to detect benzimidazole resistance, namely, the egg-hatch assay, tubulin-binding assay, and a larval-development assay, were evaluated by estimating the level of benzimidazole resistance in three field isolates of Haemonchus contortus compared with a susceptible reference strain. Comparisons were also made with estimates of benzimidazole resistance of the three field strains obtained from an in vivo controlled anthelmintic efficacy test. All three in vitro tests showed similar, consistent results which also suggested greater sensitivity than the in vivo assay. These results indicate that selection of an in vitro technique to determine benzimidazole resistance should therefore be based on considerations other than precision, such as technical expertise, availability of equipment, cost and speed in which diagnosis is required.     

T-cell receptor and immunoglobulin gene polymorphisms and resistance to Haemonchus contortus in sheep

SUMMARY: DNA extracted from animals originating from three large half-sib sheep families derived from a single, highly resistant ram were probed with ovine T-cell receptor α and β chain and immunoglobulin lambda light chain variable and constant region cDNA probes. When genomic DNA was digested with TaqI, 19 polymorphic bands were detected with the lambda light chain immunoglobulin variable region probe and 6,4 and 5 polymorphic bands were detected with the T-cell receptor α and β probes and the lambda immunoglobulin constant region probes, respectively. All animals had been phenotypically assessed for Haemonchus contortus resistance by faecal egg counts and using best linear unbiased statistical methods, no statistically significant associations were found between RFLP banding patterns detected at these loci and faecal egg counts. The results show that germline polymorphism at these loci does not account for a significant proportion of the variation in resistance to Haemonchus contortus in this flock. ZUSAMMENFASSUNG: T-Zellrezeptor und Immunoglobulin-Genpolymorphismus und Resistenz von Schafen gegen Haemonchus contortus DNS aus Tieren von drei gro?en Halbgeschwister-Schaffamilien aus einem einzelnen hochgradig resistenten Schafbock wurden mit cDNS-Sonden aus ovinen T-Zellrezeptor α- und β-Ketten und Immunoglobulin λ leichten variablen Ketten und konstanten Regionen untersucht. Nach Verdau der genomischen DNS mit TaqI 19 wurden polymorphe Banden mit der λ leichte Ketten Immunoglobulin variablen Region-Sonde entdeckt, 6, 4 und 5 polymorphe Bande wurden mit T-Zellrezeptor α- und β-Sonden und mit der Sonde der λ Immunoglobulin konstanten Region gefunden. Alle Tiere wurden ph?notypisch in Hinblick auf Haemonchus contortus-Resistenz mittels f?kaler Eiproben untersucht. Statistische Analyse mittels linearer unverzerrter Pr?diktion ergab keine statistisch signifikanten Zusammenh?nge zwischen RFLP Bandmustern und f?kalen Eizahlen. Genetischer Polymorphismus an diesen Loci scheint nicht für einen signifikanten Teil der Variabilit?t in der Resistenz gegen Haemonchus contortus verantwortlich zu sein.     

The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep

Resistance to the benzimidazole (BDZ) class of anthelmintics in nematodes of sheep has become a common and global phenomenon. The rate at which the selection process and development of resistance occurs is influenced by a number of factors. Of these, the effects of stage-specific exposures to anthelmintic were investigated with a BDZ-resistant strain of Haemonchus contortus (HCR) over five parasite generations. Sheep were infected at each generation with the HCR strain and were treated with thiabendazole (TBZ), either 5 days post-infection (p.i.) (larval line), 21 days p.i. (adult line), or left untreated (no selection line). Additionally eggs from each generation were exposed to TBZ (egg line). Geometric worm burdens were calculated from post-mortem worm counts, both at the start of the study, and after the final selection studies for each of the selection lines. Egg hatch assays (EHAs) were also conducted throughout the study. All data relating to worm burdens and EHAs for each generation were analysed by linear regression to produce dose titration curves and lethal dose(50) (LD(50)) values for each of the selection lines. Over the five generations, LD(50) values on dose-response were increased and worm survival occurred at higher dose rates of TBZ irrespective of the parasite stage exposed to treatment. A similar picture was seen with ED(50) values, which showed a fluctuating but generally upward trend for each of the three selection lines. In contrast, LD(50) and ED(50) values were decreased in the no selection line, indicating some degree of reversion albeit to levels still considered to be BDZ-resistant.     

Levels of benzimidazole resistance in Haemonchus contortus and Trichostrongylus colubriformis recorded from an egg hatch test procedure

A method of using commercially available benzimidazole anthelmintics in an egg hatch test, to record levels of resistance is described. The test shows that a side-resistance is present to all the benzimidazole anthelmintics tested against both Haemonchus contortus and Trichostrongylus colubriformis after selection with thiabendazole. Additional selection on the field resistant strain of H contortus with thiabendazole showed increased levels of resistance to all the compounds tested while similar selection of T colubriformis failed to change the level of resistance after 20 generations. The level of resistance recorded by the egg hatch test in vitro is greater than that recorded for adults in vivo for the CF strain of H contortus and T colubriformis. Problems associated with reading the test are mentioned as are the different levels for the threshold of activity.     

Trichostrongylus colubriformis and Haemonchus contortus infections in light bodyweight Merino lambs

OBJECTIVE; To show that low bodyweight is a predisposing cause of high Trichostrongylus colubriformis and Haemonchus contortus burdens and egg counts in Merino lambs. DESIGN: A comparison was made, among lambs of different bodyweights, on the effect on immunity of a primary or secondary viable infection with T colubriformis or H contortus larvae. PROCEDURE: Sixty-one Merino lambs, 1 or 6 months of age, were penned indoors and given primary, or both primary and secondary, infection of T colubriformis or H contortus. Faecal egg counts, worm counts and parasite-specific immunoglobulin concentrations were examined for their relationships with bodyweight. RESULTS: Bodyweight at the start of a primary infection was correlated with worm burden, worm fecundity and jejunal IgA antibody concentration. Merino lambs weighing less than 23 kg at the time of first exposure to T colubriformis or H contortus had impaired ability to develop protective mucosal immunity and to resist homologous challenge. CONCLUSION: If the goal is to ensure that lambs develop immunity before weaning, then every endeavour should be made to achieve the combination of critical bodyweight and exposure to moderate levels of nematode infection as soon as possible.     

Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes

In order to monitor and eventually control the spread of drug-resistant Haemonchus contortus, knowledge of the molecular mechanisms underlying resistance is essential. Here we phenotypically and genotypically characterize three multidrug-resistant H. contortus field isolates from Australia and South Africa. All were significantly less susceptible to thiabendazole than a sensitive reference strain in an in vitro egg-hatch assay. The sensitivity was further reduced in a surviving population after treatment of infected sheep with albendazole. The beta-tubulin genes were amplified from genomic DNA of the H. contortus isolates, cloned, and sequenced. There was a high degree of sequence variation. The known mutation phenylalanine-200 to tyrosine (F200Y) occurred in 60% of the sequences from resistant isolates, but not in the sensitive reference. Interestingly, 90% of the beta-tubulin sequences from resistant isolates lacking tyrosine-200 carried another mutation nearby, glutamate-198 to alanine (E198A). This mutation was not found in the sensitive isolate, nor in sequences from resistant isolates carrying the mutation F200Y. However, the mutation E198A is known from benomyl-resistant isolates of phytopathogenic fungi such as Monilinia fructicola. The finding that alanine-198 correlates with thiabendazole resistance in H. contortus isolates from South Africa and Australia suggests that also in nematodes, the mutation E198A plays a role in benzimidazole resistance. Alanine-198 alleles of beta-tubulin can be detected by PCR-RFLP and we suggest to include this test in future surveys of H. contortus field populations.     

Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes

Benzimidazole resistance is a common problem in parasitic nematodes of ruminants and early detection is vital if its spread is to be monitored and controlled. Real time PCR offers a fast and reliable method for rapid detection and measurement of resistance allele frequencies. In Haemonchus contortus a single nucleotide polymorphism at codon 200 of the beta-tubulin gene (TTC to TAC), causing a phenylalanine to tyrosine amino acid substitution, has been shown to be involved in many cases of resistance. Locked nucleic acid (LNA) Taqman probes have been used in this work to detect and measure the frequency of resistance alleles in individual and multiple H. contortus. Detection of resistant genotypes using LNA Taqman probes in individual H. contortus is simpler and more reliable than with previously described assays. Measurement of the frequency of resistant alleles in populations of H. contortus was achieved by using the cycle threshold (C(t)) values and a standard curve derived from populations with known allele frequencies. Results using the LNA probes on individual and multiple worms gave similar results to the allele specific PCR. The sensitivity of the LNA assay on multiple nematodes allowed reliable detection of > or = 10% resistance allele frequency. Using the final fluorescence method, it was possible to differentiate populations with approximately 0, 5 and 10% resistance allele frequencies.     

Changes in resistance in mixed infections of susceptible and benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis passaged in sheep

Benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis were each diluted with equal numbers of their respective susceptible genotypes and passaged in separate worm-free sheep. The progeny of the mixed susceptible and resistant infections were diluted with equal numbers of susceptible genotypes before passaging on two further consecutive occasions in worm-free sheep. In H contortus the amount of thiabendazole required to prevent a 50 per cent egg hatching (LC50) was reduced at each generation, but the reduction was significant (P less than 0.05) only at the third passage. In T colubriformis the LC50 for thiabendazole was reduced only at the first passage and thereafter remained constant. The reduction was not significant. In another experiment, the dilution with susceptible genotypes was made only at the first passage. Thereafter for two further generations the progeny produced from the mixed infection were passaged in worm-free sheep. In H contortus a reduced amount of thiabendazole was required to prevent a 50 per cent egg hatching at each generation, but did not reach a level of significance. The result for T colubriformis was identical to the continuous dilution experiment.     

捻转血矛线虫ES24抗原基因的克隆与表达特性分析

根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。     

伊维菌素耐药及敏感捻转血矛线虫国内分离株的鉴定

为解决国内开展驱虫药耐药性相关研究中虫种资源缺乏问题,该研究采用国际通用方法开展了华北地区捻转血矛线虫耐伊维菌素虫株的分离工作,并采用传统形态学方法对其进行鉴定。对来源不同的耐伊维菌素捻转血矛线虫虫株进行分离,结果显示：华北地区分离到了4株对伊维菌素6倍耐药及1株对其敏感的捻转血矛线虫。该研究将为国内开展捻转血矛线虫耐伊维菌素研究及新型驱虫药物的开发提供虫种资源。     

Reduced efficacy of ivermectin, abamectin and moxidectin against field isolates of Haemonchus contortus

OBJECTIVES: To investigate the reduced efficacy of ivermectin, abamectin and moxidectin against two field isolates of Haemonchus contortus. These isolates were identified on separate properties in the New England region of New South Wales. PROCEDURE: Reduced efficacy of macrocyclic lactone anthelmintics against two field isolates of H contortus was suspected. These isolates were obtained from sheep on separate farms and pen trials were performed to investigate the efficacy of macrocyclic lactones. The percentage efficacy was calculated for moxidectin, ivermectin and closantel against the isolate from one farm (VHR23) and for moxidectin, ivermectin and abamectin against the isolate from the second (VHR29). The persistent activity of moxidectin against both isolates was investigated. RESULTS: Ivermectin and closantel were found to have efficacies below 80% against established populations of VHR23. Moxidectin was effective against an established population of VHR23 but the persistent activity was reduced to 7 days. Moxidectin was also found to be effective against established populations of VHR29, however, ivermectin and abamectin were found to have efficacies below 80%. There was no evidence of persistent activity of moxidectin against VHR29. CONCLUSION: A reduction in efficacy of abamectin and/or ivermectin against field isolates of H. contortus was identified from two farms in the New England region of New South Wales. The persistent effect of moxidectin was reduced against both isolates.     

鸡β-actin基因实时荧光定量PCR方法的建立

根据GenBank上鸡β-actin基因的序列，在保守区域设计并合成一对引物，采用SYBR Green I染料建立了荧光定量PCR法（real-time PCR）。以常规PCR产物为标准品，建立了标准曲线，并进行了熔解曲线分析。结果表明，标准曲线Ct值检测范围为12~31,扩增效率为95.1%；熔解曲线分析结果显示产物特异的单个峰，其Tm为88±0℃，检测周期从RNA提取到荧光定量PCR结束只需4 h。本试验建立的鸡β-actin基因实时荧光定量PCR法扩增效率高、检测范围广、检测周期短，为β-actin基因作为内参基因进行鸡功能基因与病原基因表达的定量分析奠定了基础。     

模拟增温下短花针茅实时荧光定量内参基因的筛选及验证

本研究以荒漠草原植物短花针茅(Stipa breviflora)为材料,在模拟增温处理下,通过q RT-PCR试验分析9个候选内参基因的表达稳定性,运用Delta-CT法,ge Norm,norm Finder,Bestkeeper软件及Ref Finder在线工具进行候选内参基因的稳定性分析。经普通PCR剔除3个内参基因后,6个候选内参基因表达稳定性由高到低排序为:TLF,18 s r RNA,APRT,TUBα,EF-1α,Actin2。以TLF作为内参基因,在模拟增温下短花针茅转录组数据库中随机选择5个基因进行验证。q RT-PCR结果表明,5个验证基因在增温与对照之间的相对表达量存在倍率变化,且与转录组测序中的变化趋势一致,从而确定TLF为短花针茅增温处理下的最佳内参基因。     

Evaluation of a laboratory test to detect resistance to closantel in Haemonchus contortus

OBJECTIVE: To evaluate a laboratory test for closantel resistance in Haemonchus contortus. PROCEDURE: Field isolates of H contortus, known to be resistant to closantel, were tested in the assay. In addition, mixtures of closantel-susceptible and closantel-resistant laboratory reference strains were tested to develop a method of predicting the proportion of resistant worms in a sample from the field. RESULTS: The assay correctly identified as resistant all of the closantel-resistant field isolates of H contortus. It also identified one isolate with an in vivo efficacy of 98% as having emerging resistance. Testing of the mixtures of laboratory reference strains revealed that an isolate would be classified as resistant when it consists of about 25% or more resistant worms. Test samples that are not fully susceptible yet contain less than 25% resistant worms may be classified as emerging resistance. CONCLUSION: The in vitro migration assay is a sensitive method of detecting closantel resistance in H contortus.     

The efficacy of netobimin against a benzimidazole susceptible and a resistant strain of Haemonchus contortus in sheep in The Netherlands

Netobimin was tested for efficacy against Haemonchus contortus using 7 groups of 5 parasite-free lambs of six months age. The lambs in group 1 and 2 were infected with 10,000 larvae of a benzimidazole susceptible strain and those in groups 3-7 with the same dose of a resistant strain. The following treatment scheme was applied 21 days after infection: lambs in groups 2 and 4-7.5 mg kg-1 netobimin, in group 5-20 mg kg-1 netobimin, in group 6-5 mg kg-1 oxfendazole and in group 7--3.8 mg kg-1 albendazole. The lambs in groups 1 and 3 remained untreated. All lambs were slaughtered 28 days after infection. Egg counts decreased in all lambs after treatment, but increased again in lambs in groups 4, 6 and 7. There was a slight increase in lambs in group 5, while those in group 2 remained negative. Post-mortem worm counts showed a reduction of 99.8 per cent in lambs in group 2 compared to those in group 1. In lambs in group 4-7 the reduction of worm counts was respectively 40.9, 89.5, 24.7 and 40.7 per cent compared to those in group 3. Egg development assays carried out 20 days after infection showed an average LD50 of 0.46 mg ml-1 thiabendazole for the resistant strain. After treatment (day 27) the LD50 was 0.53, 0.48, 0.58, 0.56 and 0.47 in lambs in the groups 3-7. It is concluded that netobimin and other (pro)-benzimidazoles should not be used in cases of benzimidazole resistance and that levamisole, pyrantel tartrate or ivermectin are preferable.     

The anthelmintic efficacy of albendazole against Fasciola hepatica and benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

The anthelmintic efficacy of albendazole (methyl [5-(propylthio) - 1H - benzimidazole -2 -yl] carbamate) against immature and adult Fasciola hepatica and against standardised strains of benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis was evaluated in experimentally infected sheep. A single intrarumenal treatment of dose rates of 3.8 and 7.6 mg/kg was ineffective against immature (six weeks old) F hepatica. Dose rates of 5.7 and 7.6 mg/kg reduced the number of mature (12 weeks old) F hepatica by 70 and 91 per cent respectively. Dose rates of 5.7 and 7.6 mg/kg removed 92 and 99 per cent of four-week-old, benzimidazole resistant H contortus and 89 and 99 per cent of four-week-old, benzimidazole resistant T colubriformis.     

捻转血矛线虫(H.contortus)ZJ株H11蛋白基因的克隆及序列分析

参照已发表的捻转血矛线虫H11蛋白基因序列设计引物，以H．contortuss ZJ株的总RNA为模板，进行RT—PCR扩增，成功扩增出H11基因。将PCR产物与pUCm—T载体连接后，转化DH5α感受态细胞，筛选阳性克隆并测序。序列分析表明，其核苷酸序列与国外报道的H11基因的同源性为99．5％。编码氨基酸序列具有氨基肽酶的保守序列，推测可使虫体不能获得所必需的氨基酸而导致死亡。